Use of mutagenic dna polymerase for producing random mutations

ABSTRACT

The invention concerns a method for random mutagenesis comprising the replication of a DNA sequence in the presence of an efficient amount of at least a mutase, for example a Pol β, the random mutagenesis rate being at least of the order of 1 mutation for 400 base pairs. The replication product, optionally recombined and amplified, is cloned in an expression vector so as to generate mutated polypeptides which will be selected on the basis of the desired property or properties.

[0001] The present invention relates to the use of one or more mutagenic DNA polymerases for modifying DNA sequences which are at least partially coding. Such a process should make it possible to rapidly obtain a large number of random mutants, and therefore mutated polypeptide (for example protein) libraries, and makes it possible to select polypeptides (for example proteins) of interest according to a predetermined phenotype. This tool is therefore directed in particular toward any experimenter wishing to modify a protein either in the context of a fundamental study or with a view to a phenotypic improvement for industrial purposes.

[0002] Various techniques have been developed for promoting in vitro mutagenesis on DNA sequences. Among these, mention may be made of site-directed mutagenesis and random mutagenesis.

[0003] Site-directed mutagenesis is a method which consists in altering the structure of a protein, in vitro, by simple modification of targeted codons in the sequence of the DNA. Thus, amino acids can be substituted at known or supposed active sites of the protein.

[0004] Random mutagenesis in vitro consists in introducing, during a replication or recombination step, mutations distributed randomly over the sequence of a gene or of a gene fragment. The mutations can be introduced throughout the length of the coding region of a gene, or can be confined to quite specific DNA segments. Unlike site-directed mutagenesis, a precise knowledge of the structure of the protein is not necessary to carry out random mutagenesis.

[0005] Several methods of random mutagenesis have been developed.

[0006] In a first, widely implemented approach, the polymerase chain reaction (PCR) is used under conditions which promote the introduction of mutations (error-prone PCR). With Taq polymerase, the frequency of base substitution can reach 10⁻³, i.e. 1 substitution per 1 000 base pairs (Moore et al. (1996) “Direct evolution of para-nitrobenzyl esterase for aqueous-organic solvant” Nat. Biotech. 14: 458-467). In other cases, a frequency of approximately 1 substitution per 500 bases has been reached (Sweasy et al. (1993) “Detection and characterization of mammalian DNA polymerase β mutants by functional complementation in Escherichia coli” Proc. Natl. Acad. Sci. USA 90: 4626-4630; Diaz et al. (1991) “PCR-mediated chemical mutagenesis of cloned duplex DNAs”. Biotechniques 11: 204-211).

[0007] However, this technique has several drawbacks. In particular, it requires a step in which the substrate to be modified is treated with genotoxic chemical compounds. In addition, it produces too low a rate of mutagenesis. It is not therefore suitable for the construction of a library of mutants and the rapid selection of advantageous mutants.

[0008] In another approach, use is made of oligonucleotides (for example 19 or 47 base pairs) of random sequence synthesized by a chemical process (degenerate oligonucleotides), which are inserted into a gene, preferably into the region encoding the active part of the enzyme, and used to provide a diversity of proteins. (Horowitz et al. (1986) “Promoters selected from random DNA sequences” Proc. Natl. Acad. Sci. USA 83: 7405-7409; Dube et al. (1989) “Mutants generated by the insertion of random oligonucleotides into the active site of the β-lactamase gene” Biochemistry 267: 5703-5707). This procedure requires the synthesis, also expensive, of many mutated oligonucleotides.

[0009] Yet another approach, based on the use of homologous recombination, similar to the natural process of genetic mixing which takes place during evolution, can be used. This method is called shuffling (Stemmer (1994) “DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution” Proc. Natl. Acad. Sci. USA 91: 10747-10751). It consists in carrying out a PCR on fragments of a gene or fragments of several homologous genes, subsequent to a random digestion with DNAse I. The small fragments derived from this digestion serve as primers with respect to one another during the PCR, and lead to the introduction of random mutations by recombination. This method is laborious and takes place in several steps (digestion of the DNA, recombination by PCR in the presence of “mega-primers”, etc.), which involves difficulties in implementation. It has been used in improving the activities of several enzymes such as Green Fluorescent Protein, β-lactamase, and also the arsenate detoxication operon.

[0010] The fidelity of several DNA polymerases has been tested in order to measure their mutagenic capacity. Among these, the Klenow fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase, the T7 phage DNA polymerase sequenase, and the Taq DNA polymerase. Among all these polymerases, the Taq polymerase exhibits the least fidelity. However, the frequency of mutagenesis remains too low to envisage its use for random mutagenic purposes without modification of the reaction conditions or of the polymerase itself (Cadwell et al. (1992) “Randomization of genes by PCR mutagenesis” Cold Spring Harbor Laboratory 2: 28-33).

[0011] Consequently, despite the techniques developed, there remains, to date, a need for a random mutagenesis technique which is simple to implement, which does not involve the use of genotoxic chemical compounds or of complex steps, or the synthesis of numerous oligonucleotides, and which generates a sufficiently high frequency of random mutations to envisage the creation of a library of proteins.

[0012] After lengthy studies, the Applicant has demonstrated, surprisingly, that it is possible to use a mutase during the replication of a DNA, in order to obtain a sufficiently high frequency of random mutagenesis.

[0013] Document WO 98/23733 describes a method for identifying thermostable mutant polymerases having increased or decreased replication fidelity compared to a native polymerase. The possibility of using such mutant polymerases, the fidelity of which is decreased, in a method of random mutagenesis is very briefly mentioned, but no information is given, the document focusing on the identification of the mutants and on the use of the mutants having increased replication fidelity.

[0014] The invention thus relates to a method of random mutagenesis which comprises the replication of a DNA sequence in the presence of at least one mutase in effective amount. The DNA sequence may be a gene fragment or a complete gene, which can encode a protein, for example an enzyme.

[0015] Several mutases can be used, either simultaneously or successively. The mutase(s) used will preferably be thermostable. The mutases which can be used may be native polymerases, i.e. nonmutated polymerases, or optionally mutated mutases.

[0016] The term “mutase” is here intended to mean a DNA polymerase with a mutagenic level at least as high as that of DNA polymerase β (Pol β); in other words, a DNA polymerase at least as mutagenic as Pol β. Such a mutase will introduce unpaired nucleotides, a source of mutations during the replication of the DNA, in a random manner, into a gene or a gene fragment.

[0017] A suitable mutase can therefore be Pol β, or else Pol ι, Pol η or Pol κ.

[0018] DNA Pol β is a small polypeptide of 39 KD. It is an enzyme which is highly conserved in higher eukaryotes. Its primary function is thought to be the “at all costs” repair of damaged DNA, but it also has a role in the replication of native DNA. DNA Pol β is expressed at a constant level during the cell cycle, and exposure of the cells to xenobiotic agents, for example radiation, induces its expression.

[0019] To date, it has never been used in random mutagenesis. It differs from the DNA polymerases conventionally used in mutagenesis by virtue of its infidelity during DNA replication, this infidelity being thought to be related to the absence of associated corrective exonucleated activities (Kunkel T. A. (1985) “The mutational specificity of DNA polymerase-β during in vitro DNA synthesis” J. Biol. Chem. 260: 5787-5796; Kunkel, T. A. (1986) “Frameshift mutagenesis by eucaryotic DNA polymerases in vitro.” J. Biol Chem. 261: 13581-13587). Unlike these other polymerases used, which exhibit insufficient infidelity to rapidly and easily generate random mutations which can result in the creation of mutated protein libraries, the degree of infidelity of Pol β is particularly high, and can reach 10⁻².

[0020] Various sources of Pol β can be used: Hela cells (cell extracts), chicken, rat and human liver.

[0021] A native Pol β or a mutated Pol β can be used.

[0022] Whatever the mutase(s) used, the frequency of random mutagenesis is at least of the order of 1 mutation per 400 base pairs, preferably at least of the order of 1 mutation per 300 base pairs, more preferably at least of the order of 1 mutation per 200 base pairs, even more preferentially at least of the order of 1 mutation per 100 base pairs, or at least of the order of 1 per 50 base pairs.

[0023] The mutase(s) used will preferably be in the form of (a) cell extract(s).

[0024] The method according to the invention therefore comprises the replication of a DNA sequence in the presence of an effective amount of at least one mutase. Those skilled in the art will be able to define this effective amount. Due to the particular mutagenic properties of the mutase(s) used for the replication, copies of the starting DNA sequence are obtained which are not true copies, and which carry random mutations in sufficient number to generate a set of mutated polypeptides which can be used to create a polypeptide library and to select one or more polypeptides exhibiting one or more desired properties. The polypeptides can be proteins such as enzymes, of plant or animal, in particular human, origin. The desired property(properties) may, for example, be improved heat resistance, better effectiveness, a more rapid or more targeted action, binding or improved binding to a receptor, resistance or improved resistance for certain compounds, etc.

[0025] The replication step can be followed by a recombination step. This recombination can be a step of digestion of DNA followed by a PCR amplification step. In addition, during the PCR, the hybridization and polymerization steps can be combined in a single, very short step. The recombination can also be a step of digestion of DNA followed by a step of ligation of the digestion products.

[0026] The PCR, when it is used, therefore simply serves to amplify the material obtained at the end of the mutagenesis step. Preferably, use is then made of amplification primers the length of which is such that they require an amplification temperature of at least 70° C., which improves the specificity of the PCR.

[0027] The replication product, which carries (a) random mutation(s), and which is optionally recombined and amplified, can then be cloned into an expression vector in order to generate the mutated polypeptides, which are isolated. These mutated polypeptides will be grouped together in a polypeptide library, and may be selected as a function of the desired property or properties.

[0028] The mutagenic properties of the mutase(s) used may be further increased, in order to further increase the number of mutations generated. Several techniques are possible.

[0029] Thus, in one embodiment of the method according to the invention, magnesium is substituted with manganese or cobalt in the PCR reaction (Beckman et al. (1985) “On the fidelity of DNA replication: manganese mutagenesis in vitro.” Biochemistry 24: 5810-5817).

[0030] Another embodiment of the method according to the invention comprises the use of a biased nucleotide pool, i.e. of a pool of nucleotides in which one or more nucleotides are favored relative to the others. By way of example, use may be made of a biased pool in which three of the natural nucleotides are favored relative to the fourth (Cadwell et al. (1992) “Randomization of genes by PCR mutagenesis.” Cold Spring Harbor Laboratory 2: 28-33).

[0031] In another embodiment, one or more mutagenic nucleotide analogs are incorporated. Such an analog may be 8-oxoguanine (Zaccolo et al. (1996) “An approach to random mutagenesis of DNA using mixture of triphosphate derivatives of nucleoside analogues” J. Mol. Biol. 255: 589-603).

[0032] Use may also be made of another DNA polymerase in combination with the mutase(s), whether simultaneously or successively.

[0033] Finally, it is possible simply to modify one or more parameters of the reaction medium, such as the pH, the temperature or the salt concentration, in order to modify the action of the mutase(s). The present application is also directed toward any DNA and any polypeptide or protein obtained, or which can be obtained using a method according to the invention. It is in particular directed toward any library of DNA, of polypeptides or of proteins obtained, or which can be obtained by collection of DNA, polypeptides or proteins according to the invention. It is also directed toward any method for identifying a compound exhibiting an activity or property of interest, in particular in the medical, agrofoods or pharmaceutical field, characterized in that it comprises identifying, in a library according to the invention, a compound exhibiting said particular activity or property.

[0034] In the following examples, reference is made to the following figures:

[0035]FIG. 1 shows very schematically the steps of the method of producing random mutations, applied to an 85-mer polynucleotide,

[0036]FIGS. 2A and 2B illustrate the results of migrations of the products thus obtained,

[0037]FIG. 3 shows the spectrum of mutations thus engendered by polymerization of the 85-mer matrix,

[0038]FIG. 4 shows schematically the production of random mutations in the lacZ gene carried by a plasmid vector,

[0039]FIG. 5 shows very schematically the steps of a method of producing random mutations by replication of the lacZ gene encoding the α-peptide of β-galactosidase, carried by the plasmid pUC18, and

[0040]FIG. 6 illustrates the distribution of the mutations thus engendered on the gene encoding the alpha-peptide of the lacZ gene, under standard conditions of replication with Pol β (all the dNTPs at 100 μm)

EXAMPLE 1

[0041] In vitro production of random mutations by replication of an 85-mer polynucleotide with DNA Pol β and use of a biased pool of deoxynucleotides.

[0042]FIG. 1 shows very schematically the various steps of the method, from the polynucleotide to the sequencing of the recombinant clones:

[0043] Material: 3′primer: 5′ GATTGAATTCCTCATTATGG 3′ (SEQ ID NO: 1) 5′primer: 5′ TGAATACTGTATGATAATCG 3′ (SEQ ID NO: 2) 85-mer matrix: 5′ TGAATACTGTATGATAATCGTGAGGATCCCGCA (SEQ ID NO: 3) TATAAGCTTTTCGATCGCCTGCAGTAACTCCACCAT AATGAGGAATTCAATC 3′

Culture Media

[0044] Complete LB medium: 1% tryptone, 0.5% yeast extract, 1% Nacl, H₂O qs 1 l. 15 g of agar are added to 1 liter of LB to prepare the LB/agar medium.

[0045] SOC medium: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose.

Bacterial Strain and Vectors

[0046] TABLE 4 Strains Genotypes Sources TOP 10 F⁻ mcrA Δ(mrr-hsdRMS-mcrBc) Invitrogen Φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ (ara-leu) 7697 galU galK rpsL (Str^(R)) endA1 nupG BL21 F⁻ ompT gal (dcm) (lon) hsdS_(B) BioLabs (r_(B) ⁻ m_(B) ⁻) with DE3, has λ prophage carrying the T7 RNA polymerase gene

In Vitro Replication of the Matrix with DNA Pol β Preparation and Purification of DNA Pol β

[0047] An overnight culture of the BL21 strain transformed with a plasmid expressing a Pol β: (His)₆ fusion protein under the control of a T7 promoter is diluted to {fraction (1/50)} in LB medium+50 μg/ml Kanamycin, and incubated at 37° C. with shaking. At OD 0.6-1, 1 mM IPTG is added and the incubation is continued for 4 hours. The cells are centifuged and then lysed with lysosyme at 100 μg/ml and 0.1% triton X100. The lysis is carried out at 30° C. for 30 min. Next, the cells are sonicated and then centrifuged. The Pol β: (His)₆ fusion is purified on a nickel column and eluted with imidazole using the resin and the buffers of the Novagen purification kit.

[0048] Labeling of the 20-mer primer:

[0049] 60 ng of the 5′ primer are labeled with 10 units of T₄ polynucleotide kinase (New England BioLabs) for one hour at 37° C. in a buffer containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 5 mM dithiotreitol and 20 μCi ³²P-γ-ATP. The enzyme is then inactivated by incubating the reaction mixture at 70° C. for 10 minutes.

[0050] Hybridization of the labeled primer to the matrix 60 ng of the 5′ primer labeled with ³²P-γ-ATP are added to 300 ng of the matrix in a hybridization buffer (100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂). The mixture is first denatured at 70° C. for 10 minutes. Hybridization of the two oligonucleotides is then obtained by continuing the incubation until the temperature of the bath has reached ambient temperature.

[0051] Replication of the Matrix

[0052] One unit of DNA Pol β is added to 5 ng of the 85-mer matrix hybridized to 20 ng of 20-mer oligonucleotide labeled with ³²P-γ-ATP, in a reaction mixture containing 25 mM HEPES (pH 8.5), 125 mM NaCl, 5 mM MgCl₂, 200 μM dATP, 200 μM dGTP and 200 μM dTTP. These various mixtures are distributed into five tubes to which various concentrations of dCTP (0 dCTP, 0.2 μM dCTP, 2 μM dCTP, 20 μM dCTP, 200 μM dCTP) are added. After incubation for one hour at 37° C., part of the mixture is loaded onto an acrylamide gel (15% acrylamide, 7 M urea, 30% formamide) in order to control the reaction products. The other part is supplemented with dCTP at a final concentration of 200 μM and the reaction is continued for one hour. Finally, the reaction is stopped with the stop buffer (90% formamide/0.1% xylene cyanol/0.1% bromophenol blue/0.1 mM EDTA). The samples are then denatured for 10 minutes at 70° C. and loaded onto an acrylamide gel in order to visualize the replication products. These products are revealed by exposure on an autoradiographic film.

[0053] Polymerase Chain Reaction (PCR)

[0054] The PCR reaction was carried out by adding 1 μl of the replication product obtained by the action of Pol β to a mixture containing the polymerization buffer (20 mM Tris HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Triton) (New England Biolabs), 1.5 mM MgSO₄, 200 mM dNTP, 20 pmol of each 5′ and 3′ oligonucleotide, and 5 u of high fidelity Vent polymerase (New England Biolabs). These mixtures were incubated in a BioRad thermocycler according to the following program: (95° C, 5 min)-(95° C. 30 sec.-50° C. 30 sec.-72° C. 30 sec.)×30 cycles-(72° C.-5 min.). The PCR products are then loaded onto a 1% TAE agarose gel. After migration and staining of the gel with ethidium bromide, the 85-mer band corresponding to the product of amplification of the matrix is visualized by trans-illumination with ultraviolet light.

[0055] Cloning

[0056] The cloning of the amplification products was carried out using the Zero Blunt TOPO PCR Cloning Kit from Invitogen. 1 μl of amplification product obtained by PCR is added to 10 ng of the cloning vector pCR-Blunt II-TOPO (Invitrogen) contained in the following buffer: 50% glycerol, 50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 100 μg/ml BSA. The mixture is maintained at ambient temperature for five minutes and then incubated at 4° C. 2 μl of the cloning reaction are then added to Top 10 competent cells (Invitogen), and incubated for 30 minutes at 4° C. A heat shock is carried out at 42° C. for 30 seconds. The cells are then again placed on ice before being incubated at 37° C. for one hour in 250 μl of SOC medium. Finally, the transformants are selected on LB/agar medium containing 50 μg/ml of kanamycin.

[0057] Extraction of the Plasmidic DNA

[0058] The recombinant plasmids are extracted and purified with the Quia Prep Spin system from Quiagen. The plasmids are eluted in 50 μl of sterile water and controlled on a gel of 1% agarose in 1×TAE, after staining with ethidium bromide and illumination by ultraviolet radiation.

[0059] Sequencing

[0060] The sequencing reaction is carried out with the SequiTherm EXCEL II DNA sequencing Kits-LC system from Epicentre Technologies. 1 pmol of M13 primer labeled with IRD700 (MWG Biotech) and 300 ng of recombinant plasmids are incubated with 1U of Sequitherm EXCEL II DNA polymerase in the Sequitherm EXCEL II sequencing buffer (Epicentre Technologies). This mixture was incubated in a PTC-100 thermocycler (MJ Research, Inc.) according to the following program: (95° C. 5 min.)-(95° C. 30 sec.-57° C. 15 sec.-70° C. 1 min.)×30 cycles-(70° C.-10 min.). The reaction products are then loaded onto a sequencing gel and analyzed using a LONG READIR 4200 sequencer (Li-COR).

[0061]FIG. 2 shows the result of the migration of the products of replication with Pol β. Part A represents the results after replication for one hour with the various concentrations of dCTP (1=0 μM, 2=0.2 μM, 3=2 μM, 4=20 μM, 5=200 μM) and 200 μM of each of the other dNTPs (dATP, dGTP, dTTP). In part B, lanes 6, 7, 8 and 9 represent the products of replication 1, 2, 3 and 4, respectively, after complementation of dCTP at a concentration of 200 μM and continuation of the reaction for one hour.

[0062] Table 1 below gives a summary of the production of random mutations by DNA Pol β on an oligonucleotide. It comprises the frequencies of the mutations generated by replication with DNA Pol β under standardized conditions and using a biased pool of deoxyribonucleotides. TABLE 1 Sequenced Mutation dCTP dATP dGTP dTTP clones frequencies 200 μM  200 μM 200 μM 200 μM 26 0.9 × 10⁻² 20 μM  200 μM 200 μM 200 μM 26 1.2 × 10⁻² 2 μM 200 μM 200 μM 200 μM 27 1.36 × 10⁻²  0.2 μM   200 μM 200 μM 200 μM 20 1.7 × 10⁻² 0 μM 200 μM 200 μM 200 μM 18 1.8 × 10⁻²

[0063] The experiment under standardized conditions of polymerization with Pol β (the 4 dNTPs are present at a concentration of 200 μM) shows a high mutation frequency, approximately one mutation every 100 nucleotides replicated, thus confirming the mutagenic capacity of this polymerase lacking fidelity.

[0064] Pol β is capable of completely replicating the 85-mer matrix, even in the absence of one type of nucleotide out of four. These replication reactions result in an increase in the rate of mutations which is inversely proportional to the concentration of the fourth nucleotide dCTP (table 1). This confirms the potentialization of the mutagenic capacity of Pol β by the combination of a nucleotide bias in the reaction medium.

[0065] Even in the absence of a biased pool of nucleotides, the frequency of the mutations observed is 0.9×10⁻², that is to say close to 1 per 100 base pairs.

[0066] In addition, these mutations are distributed throughout the length of the matrix, and in a random manner. The mutagenesis is not therefore confined to specific sites.

[0067]FIG. 3 represents the spectrum of mutations engendered by the polymerization of the 85-mer matrix with Pol β under the following conditions: 200 μM of DATP, dGTP and dTTP, and 0 μM of dCTP.

EXAMPLE 2

[0068] In vitro production of random mutations in the lacZ gene carried by a plasmid vector.

[0069] The aim of this experiment is to perform SV40 replication of a pBK-CMV matrix (containing the SV40 origin, the Neo^(r)-Kan^(r) double resistance gene and the sequence encoding the α-peptide of β-galactosidase allowing α-complementation in Δα-lacZ strains) with Hela extracts and the T antigen, and to add purified rat DNA Pol β during the experiments. The difference in frequency of mutagenesis induced by the excess of Pol β is measured using the mutagenesis test described below (white/blue screen).

[0070] Bacterial Strains, Plasmids, Restriction Enzymes

[0071] The plasmid pBK-CMV (Strategene) was amplified in a dam⁺ bacterium (DH5α) and purified using the Wizard Plus DNA Purification System (Promega). The DpnI enzyme comes from BioLabs. TABLE 5 Strains Genotypes Sources MC1061Mut S F⁻ araD139 Δ(araleu)7696 galE15 T. Kunkel, galk16 ΔlacX74 rpsL (Str^(R)) hsdR2(r_(K) ⁻ USA m_(K) ⁺) mcrA mcrB1 [mutS : :Tn10] JM109 F′ traD36 lacI^(q) Δ(lacZ)M15 T. Kunkel, proA + B + /e14⁻ (McrA⁻) Δ(lac-proAB) USA thi gyrA96 (NaI^(r)) endA1 hsdR17 (r_(K) ⁻m_(K) ⁺) relA1 supe44 DH5α EndA1 hsdR17 (r_(K) ⁻m_(K) ⁺) supE44 thi-1 BioLabs recA1 gyrA (NaI^(r)) relA1 Δ(lacIZYA- argF)U169 deoR (φ80dlacΔ(lacZ)M15)

Preparation of the Replicative Cell Extracts Derived from Hela Cells

[0072] The cell extracts were prepared according to the procedure developed by Robert et al. (Robert et al. (1993) “Chromosome and gene analysis” Methods in Molecular Genetics. Adolph, K. W. Ed. 2: 295-313). Hela cells in suspension are cultivated in 4 liters of RPMI1640 supplemented with 9% serum and Pen/Strep5500, harvested in the exponential phase, and centrifuged at 1 500 rpm for 5 min. at ambient temperature. They are washed in 200 ml of 1×PBS at 4° C. and centrifuged at 1 500 rpm for 5 min. at 4° C. The cells are washed in 25 ml of an isotonic medium at 4° C. (20 mM Hepes-KOH, pH 7.5, 5 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 250 mM sucrose) and centrifuged at 4 000 rpm for 10 min. at 4° C. They are taken up in hypotonic medium at 4° C. without sucrose (20 mM Hepes-KOH, pH 7.5, 5 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT) and then centrifuged. They are then lysed in a potter homogenizer after adding 50 μl of aprotinin at 1.5 mg/ml, and then centrifuged at 10 000 rpm for 20 min at 4° C. The supernatant is then centrifuged at 50 000 rpm for 60 min at 4° C., and then aliquoted and frozen in liquid nitrogen. The amount of proteins present is assessed according to the

[0073] Bradford method, using BSA to establish the standard calibration range.

[0074] SV40 Replication

[0075] This replication step was carried out using the assay developed by Robert et al. The SV40 replication reaction is carried out at 37° C. for 6 h in a final reaction volume of 25 μl containing 30 mM HEPES-KOH, pH 7.8/7 mM MgCl₂/0.5 mM DDT/200 μM CTP, GTP, UTP/4 mM ATP/100 μM dCTP, dGTP, dTTP/40 mM creatine phosphate/100 μg/ml creatine phosphokinase/100 ng pBK-CMV/400 μg of Hela replicative extracts/0.5 μg Large T Antigen to which are added +/−5 μg (i.e. 1.2 U) of Pol β. The replication reaction is stopped by adding 25 μl of a buffer containing 2 mg/ml PK, 50 mM EDTA and 2% SDS, and incubating at 55° C. for 60 min. The DNA is purified by extraction with phenol, phenol/chloroform/isoamile alcohol, and ether, and then precipitated with 2.5 V 100% ethanol, 0.1 V sodium acetate and 1 μg of glycogene, and centrifuged at 13 000 rpm for 30 min. at 4° C. The DNA is then washed with 70% ethanol, centrifuged (13 000 rpm, 15 min., 4° C.) and digested with 10 U of DpnI for 2 h in order to remove any pBK-CMV matrix which has not been replicated at least once by the Hela extracts. It is finally reprecipitated and taken up in 10 μl of H₂O.

[0076] Analysis of the Mutagenesis

[0077] 2 μl of replicated plasmid are electroporated into the strain MC1061MutS, which is deficient for the mismatch repair system, in order to fix the mutations, and then the plasmid is amplified by selecting the electroporated population with Kanamycin (50 μg/ml) on a total volume of 20 ml of LB. The plasmid is extracted using the Wizard Plus DNA Purification System (Promega) and then 100 ng is electroporated into the strain JM109 allowing α-complementation. A suitable volume of the bacterial population (containing approximately 2 000 to 3 000 clones) is then inoculated into 7 ml of soft LB containing 1.6 mg of X-Gal, 1.6 mg of IPTG and 350 μg of Kanamycin, and then plated out onto dishes 14 in diameter containing 40 ml of LB/agar with 50 μg/ml of Kanamycin. The frequency of mutagenesis present on the replicated plasmid is deduced from the number of white colonies compared to the number of total colonies present on the dish.

[0078]FIG. 4 shows the summary of this production of random mutations.

[0079] Table 2 below shows the results of the production of random mutations. TABLE 2 Colonies examined DNA substrate Total Mutants Mutations × 10⁻³ Nonreplicated DNA 28 640 184 6.4 (no (AgT) DNA replicated by 141 390 918 6.5 cell extracts without Pol β DNA replicated by 209 762 2 953 14 cell extracts with Pol β

[0080] It shows that, under physiological conditions, i.e. in the presence of cell extracts, Pol β is capable of inducing random mutations on a mutagenesis target carried by a plasmid replication substrate. Once again, it is immediately noted that the frequency of the mutations induced is high, in this case 1.4 10⁻², i.e. more than 1 per 100 base pairs.

EXAMPLE 3 Replication of the lacZ Gene by Pol β and Comparison with the Results Obtained by PCR with a taq Polymerase

[0081] This example shows that Pol β introduces a frequency of mutagenesis 50 times higher than that obtained with taq polymerase by PCR.

[0082] Random mutations were produced in vitro by replication of the lacZ gene encoding the α-peptide of β-galactosidase, carried by the plasmid pUC18 (invitrogen), with DNA polymerase β, and use of a biased pool of deoxynucleotides. FIG. 5 shows very schematically the various steps of the method followed, from the replication of the lacZ gene with the polymerase β to the sequencing of the mutant clones.

[0083] Material: (SEQ ID NO: 4) 5′primer: 5′ CGCGACTCATGCGACGCATTACGAATTCGAGCTCGGTAC 3′ (SEQ ID NO: 5) 3′primer 5′ CACTCGACGCTGATGCAGTGCACCATATGCGGTGTG 3′

[0084] The parts underlined are the sequences complementary to the sequences in the 5′ and 3′ positions flanking the lacZ gene carried by the plasmid pUC18.

Culture Media

[0085] The composition of the Medium is Detailed in Example 1.

Bacterial Strain

[0086] The genotype and the origin of the strain Top 10 are described in example 1.

In Vitro Replication of the lacZ Gene with DNA Polymerase β

[0087] 1 μg of plasmid pUC18 is added to 10 pmol of the 5′ and 3′ primers in 15 μl of replication buffer (50 mM Tris-HC1, pH 8.8, 10 mM MgCl₂, 100 mM KCl, 0.4 mg/ml BSA, 1 mM DTT, 10% glycerol, Trevigen). The mixture is denatured for 5 min. at 90° C. and then hybridized for 2 min. at 55° C., before being conserved at 4° C. A solution of 15 μl of the same buffer containing 4 units of polymerase β (Trevigen), 0.5 mM Mn2+ and 100 μM of each of the 4 natural deoxynucleotides is then added to the 15 μl of hybridization mixture. The reaction of replication by polymerase β is then carried out at 37° C. for one hour. In other polymerization reactions, the pool of the 4 deoxynucleotides is biased by a lower concentration of one of the 4 deoxynucleotides compared to the three others. The replication products are then extracted with phenol-chloroform.

Selective PCR

[0088] The selective PCR reaction is carried out by adding 1 μl of the replication product (diluted to {fraction (1/10)}), obtained by the action of the polymerase A, to a mixture containing the PCR buffer (20 mM Tris HCl pH 8.4, 50 mM KCl) (Life Technologies), 1.5 mM MgCl₂, 10 pmol of the 5′ and 3′ primers, 200 μM of the 4 dNTPs and 1.25 U PLATINUM Taq DNA polymerase (Life Technologies). This mixture is incubated in a thermocycler (Eppendorf) according to the following program: (95° C., 5 min.)-(94° C., 15 sec.-55° C., 30 sec.-72° C., 30 sec.)-(94° C., 15 sec.-72° C., 30 sec.)+30 cycles-(72° C., 10 min.). This program makes it possible to specifically amplify the DNA fragments synthesized by the polymerase β.

[0089] The PCR products are then extracted with phenol-chloroform, precipitated with ethanol, and recovered in TE (10 mM Tris HCl pH 8, 1 mM EDTA).

Cloning of the PCR Products

[0090] Before being cloned into pUC18, the PCR products are digested with 20 U of NdeI (BioLabs) and 10 U BamH1 (Q-BIOgene) in a digestion buffer containing (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT and 100 μg/ml BSA) for one hour at 37° C. The digestion product is then purified with the GenClean2 kit (BIO 101) after migration on agarose gel and excision of the band containing the PCR product. The PCR product is then cloned into pUC18 (predigested with NdeI and BamH1) subsequent to a ligation (16° C. overnight) with 40 U of T4 DNA ligase (BioLabs) in a buffer containing (50 mM Tris HCl pH 7.5, 10 MM MgCl₂, 10 mM DTT, 1 mM ATP and 25 μg/ml BSA). The ligation product is then precipitated with ethanol and taken up in 5 μl of TE (10 mM Tris HC1 pH 8, 1 mM EDTA). 2 μl of the ligation reaction are then added to the Top 10 competent cells (Invitogen) and incubated for 30 minutes at 4° C. A heat shock is carried out at 42° C. for 30 seconds. The cells are then again placed in ice before being incubated at 37° C. for one hour in 250 μl of SOC medium. Finally, the transformants are selected on LB/agar medium containing 100 μg/ml of ampicillin and 60 μg/ml of X-Gal (5-bromo-4-chloro-3-indolxyl-beta-D-galactopyranoside, Euromedex). The frequency of mutants is deduced from the number of white colonies compared to the number of total colonies present on the dish.

[0091]FIG. 5 shows the summary of this production of random mutations.

[0092]FIG. 6 illustrates the distribution of the mutations thus engendered on the gene encoding the alpha-peptide of the lacZ gene, under the standard conditions for replication with Pol β (all the dNTP at 100 μM).

[0093] Table 3 below shows a summary of production of random mutations. TABLE 3 Mutation Clones frequency screened Mutants (%) Mutations/kb Replication by Taq 2 908 14 0.48 ND (100 μM dNTPS) Replication by Pol β 764 187 24.48 17.09 (100 μM dNTPs) Replication by Pol 1 749 730 41.74 18.47 β (100 μM dATP, 100 μM dGTP, 100 μM dCTP, 20 μM dTTP) Replication by Pol 1 734 1 078 62.17 23.6 β (100 μM dGTP, 100 μM dCTP, 100 μM dTTP, 20 μM dATP)

[0094] This summary shows that, under normal conditions, i.e. when the replication with polymerase β is carried out with 100 μM of each of the four dNTPs, the polymerase β is capable of producing random mutations on the gene encoding β-galactosidase. This rate of mutation (24.48%), compared to that produced by PCR amplification using the taq polymerase (Life Technologies) (0.48%), is 50 times higher. This very high rate of mutation produced by polymerase β makes it possible to screen a smaller number of mutants with the aim of isolating a mutant of a protein whose activity is enhanced or weakened. This frequency of mutants is even higher when the concentration of one of the four dNTPS is modified. For example, when the dTTP or the dATP is used separately in two independent reactions at a concentration of 20 μM, the frequency of mutants obtained is 41.7% and 62.17% respectively. This increase in frequency of mutants makes it possible to reduce even further the number of mutants to be screened.

[0095] The results of these various experiments demonstrate the relevance of using this enzyme, which is in particular native, for the purpose of creating random mutants, and make it possible to envision designing a tool based on the use of Pol β or of cell extracts/Pol β for the purpose of modifying any sequence carried by a plasmid vector. 

1. A method of random mutagenesis comprising the replication of a DNA sequence in the presence of an effective amount of at least one mutase.
 2. The method as claimed in claim 1, characterized in that the mutase is a DNA polymerase β, ι, η or κ.
 3. The method as claimed in claim 1 or 2, characterized in that the mutase(s) is (are) used in the form of (a) cell extract(s).
 4. The method as claimed in any one of claims 1 to 3, characterized in that several mutases are used, simultaneously or successively.
 5. The method as claimed in any one of claims 1 to 4, characterized in that a DNA polymerase is used in combination with the mutase(s).
 6. The method as claimed in any one of claims 1 to 5, characterized in that the frequency of random mutagenesis is at least of the order of 1 mutation per 400 base pairs, preferably at least of the order of 1 mutation per 300 base pairs, more preferably at least of the order of 1 mutation per 200 base pairs, even more preferentially at least of the order of 1 mutation per 100 base pairs, or at least of the order of 1 per 50 base pairs.
 7. The method as claimed in any one of claims 1 to 6, characterized in that the replication step is followed by a recombination step, such as a step of digestion of DNA followed by a PCR amplification step in which the hybridization and polymerization steps can be combined in a single, very short step, or else a step of digestion of DNA followed by a step of ligation of the digestion products.
 8. The method as claimed in any one of claims 1 to 7, characterized in that a biased nucleotide pool and/or one or more mutagenic nucleotide analogs are used in the replication step.
 9. The method as claimed in claim 8, characterized in that manganese or cobalt is used during the PCR.
 10. The method as claimed in any one of claims 1 to 9, characterized in that the replication product, which is optionally recombined and/or amplified, is cloned into an expression vector in order to synthesize mutated polypeptides, which are isolated.
 11. The method as claimed in claim 10, characterized in that the mutated polypeptides exhibiting the desired property or properties are selected.
 12. A DNA sequence, characterized in that it can be obtained using the method as claimed in any one of claims 1 to
 9. 13. A polypeptide, characterized in that it can be obtained using the method as claimed in claim 10 or
 11. 